Plant Physiol. (1998) 117: 859-867
Characterization of a Red Beet Protein Homologous to the
Essential 36-Kilodalton Subunit of the Yeast V-Type ATPase1
Cynthia Bauerle*,
Catherine Magembe2, and
Donald P. Briskin
Biology Department, Hamline University, 1536 Hewitt Avenue, St.
Paul, Minnesota 55104 (C.B., C.M.); and Department of Crop Sciences,
1201 West Gregory Drive, University of Illinois, Urbana, Illinois 61801 (D.P.B.)
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ABSTRACT |
V-type
proton-translocating ATPases (V-ATPases) (EC 3.6.1.3) are electrogenic
proton pumps involved in acidification of endomembrane compartments in
all eukaryotic cells. V-ATPases from various species consist of 8 to 12 polypeptide subunits arranged into an integral membrane proton pore
sector (V0) and a peripherally associated catalytic sector
(V1). Several V-ATPase subunits are functionally and
structurally conserved among all species examined. In yeast, a 36-kD
peripheral subunit encoded by the yeast (Saccharomyces cerevisiae) VMA6 gene (Vma6p) is required for
stable assembly of the V0 sector as well as for
V1 attachment. Vma6p has been characterized as a
nonintegrally associated V0 subunit. A high degree of
sequence similarity among Vma6p homologs from animal and fungal species
suggests that this subunit has a conserved role in V-ATPase function.
We have characterized a novel Vma6p homolog from red beet (Beta
vulgaris) tonoplast membranes. A 44-kD polypeptide cofractionated with V-ATPase upon gel-filtration
chromatography of detergent-solubilized tonoplast membranes and was
specifically cross-reactive with anti-Vma6p polyclonal antibodies. The
44-kD polypeptide was dissociated from isolated tonoplast preparations by mild chaotropic agents and thus appeared to be nonintegrally associated with the membrane. The putative 44-kD homolog appears to be
structurally similar to yeast Vma6p and occupies a similar position
within the holoenzyme complex.
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INTRODUCTION |
V-ATPases are electrogenic proton pumps involved in acidification
of endomembrane compartments in all eukaryotic cells (for review, see
Finbow and Harrison, 1997
). V-ATPases appear to be responsible for
acidification of vacuoles, lysosomes, Golgi cisternae, secretory
vesicles, and clathrin-coated vesicles. In addition, V-ATPases are
associated with the plasma membrane of specialized mammalian cell
types. The yeast (Saccharomyces cerevisiae) V-ATPase maintains an acidic environment in the vacuolar lumen and generates a
proton gradient that drives the transport of ions such as
Ca2+ and basic amino acids across the vacuolar
membrane (for review, see Nelson and Klionsky, 1996). In plant cells
the V-ATPase operates in conjunction with a proton-translocating
pyrophosphatase to maintain a proton gradient across the tonoplast that
supports vacuolar uptake of K+,
Ca2+, sugar, and other small metabolites by
secondary transport systems (Taiz, 1992).
V-ATPases from various species consist of 8 to 12 polypeptide subunits
arranged in an integral membrane proton pore sector (V0) and a peripherally associated catalytic
sector (V1) (Finbow and Harrison, 1997). Thus,
V-ATPases display a bipartite structure similar to mitochondrial
F1F0-ATPases. In yeast,
combined biochemical and genetic approaches have identified 10 subunits
that range from 14 to 100 kD and are required for holoenzyme assembly
and ATPase activity (Table I). The
V1 sector is comprised of 69-, 60-, 54-, 42-, 32-, 27-, and 14-kD polypeptides. The membrane V0
sector consists of 100- and 17-kD integral membrane proteins and a
tightly associated 36-kD peripheral polypeptide (Bauerle et al., 1993).
Highly purified preparations of V-ATPase from red beet (Beta
vulgaris) tonoplast contain at least 9 polypeptides, of which the
67-, 55-, 52-, 44-, and 32-kD subunits are reported to be peripherally
associated (Parry et al., 1989).
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Table I.
Subunit composition of V-ATPase from yeast and red
beet
V1 and V0 subunits of V-ATPase from yeast
and red beet. Subunits are identified based on observed molecular
mass. Numbers in parentheses indicate the 36-kD yeast and 44-kD beet
subunits examined in this study.
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Many V-ATPase subunits are functionally and structurally conserved
among all species examined (Finbow and Harrison, 1997). The
V1 70-kD catalytic and 60-kD regulatory subunits
share as much as 60 to 70% identity among diverse species. The 17-kD
V0 proteolipid is among the most highly conserved
proteins known, displaying greater than 65% sequence identity among
all species examined. This high degree of sequence similarity for
multiple subunits of the enzyme reflects the functional importance of
this ubiquitous eukaryotic proton pump.
In yeast, the 36-kD V0 subunit is encoded by the
VMA6 gene (Bauerle et al., 1993). The VMA6 gene
product, a 36-kD subunit, Vma6p, is required for V-ATPase activity as
well as stable holoenzyme assembly. In vma6 mutants lacking
the 36-kD subunit, the V0 sector does not stably
assemble, and V1 particles are unable to
associate with the membrane. Biochemical studies revealed that Vma6p is peripherally rather than integrally attached to the vacuolar membrane and thus represents a novel class of peripheral
V0 subunits. A high degree of sequence similarity
among Vma6p homologs from various species suggests that this subunit
has a conserved role in V-ATPase function (Fig.
1). Subunit homologs from fungal, insect,
and mammalian species share 41 to 57% primary sequence identity
with Vma6p. In particular, four highly conserved domains display more
than 60% identity, corresponding to residue nos. 96 to 133, 172 to 193, 212 to 227, and 314 to 322 in the yeast sequence.
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| Figure 1.
Amino acid sequence alignment of Vma6p homologs.
Dashes indicate spaces added to optimize alignment, periods indicate
amino acid identity among all seven sequences examined, and asterisks indicate stop codons. Overall homologies with Vma6p are: S. cerevisiae (S cer), 100% (Bauerle et al.,
1993); Neurospora crassa (N cras), 57%
(Melnik and Bowman, 1996); Dictyostelium discoideum
(D disc), 49% (Temesvari et al., 1994); Manduca
sexta (M sex), 47% (Merzendorfer et al.,
1997a); Bos taurus (B taur), 46% (Wang
et al., 1988); Homo sapiens (H sap), 41%
(van Hille et al., 1993); Mus musculus (M
musc), 47% (unpublished, GenBank accession no.
U21549).
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Although gene homologs of yeast VMA6 have been isolated from
various fungal and animal sources, no full-length gene homologs have
been reported from any plant source. A search of the Arabidopsis gene-fragment database (using BLAST) identified sequences bearing strong homology to yeast VMA6. By aligning overlapping
sequence fragments, we were able to assemble a partial sequence
corresponding to a putative Arabidopsis VMA6 homolog (Fig.
2). The deduced primary sequence aligned
with Vma6p residue nos. 76 to 345 and displayed all four highly
conserved domains (67, 86, 69, and 57% identity with the yeast Vma6p
sequence, respectively).
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| Figure 2.
Amino acid sequence alignment of Vma6p and deduced
partial composite sequence from Arabidopsis (A thal).
Four highly conserved regions shared between yeast and Arabidopsis
sequences are underlined. The sequence homology is 36% in the
overlapping region (corresponding to Vma6p residue nos. 76-345).
S cer, S. cerevisiae.
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The existence of a putative VMA6 homolog in Arabidopsis
hints that homologs of this essential yeast subunit may exist in other plant species. Thus, we sought to identify potential Vma6p subunit homologs in V-ATPase-enriched membrane preparations. Here we report the
initial characterization of a novel Vma6p homolog from red beet
tonoplast membranes. This 44-kD polypeptide was directly identified by
immuno-cross-reactivity with antibodies raised against yeast Vma6p.
Preliminary characterization suggests that the putative homolog is a
peripheral subunit of the V-ATPase that is tightly associated with
the membrane.
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MATERIALS AND METHODS |
Alkaline phosphatase-conjugated antibodies and Kaleidoscope
protein molecular mass standards were
purchased from Promega. Ready Gels for the MiniProtean II gel system
were from Bio-Rad. Nitrocellulose membrane was from Schleicher & Schuell. The BCA reagent kit was from Pierce. All other reagents were
from Sigma.
Preparation of rabbit polyclonal antiserum against the yeast
(Saccharomyces cerevisiae) 36-kD subunit has been described
(Bauerle et al., 1993). Corresponding preimmune serum was collected
from the same animal immediately prior to antigen exposure. Rabbit polyclonal antisera prepared against red beet (Beta
vulgaris) tonoplast V-ATPase 67- and 57-kD subunits were a
generous gift from Dr. Ron Poole (McGill University, Montreal, Quebec,
Canada).
Strains and Culture Conditions
Isogenic yeast strains SEY6211a VMA6 and SEY6211a
vma6::LEU2 have been described (Bauerle et al.,
1993). Yeast cultures were grown at 30°C with vigorous shaking in 1%
yeast extract, 2% Bactopeptone, 2% dextrose buffered at pH 5.0 with
50 mM phosphate/succinate.
Protein Sample Preparation, SDS-PAGE, and Immunoblot
Analysis
Whole yeast cell lysates were prepared in sample buffer (8 M urea, 5% SDS, 1 mM EDTA, 50 mM
Tris-HCl, pH 6.8, and 5% 
-mercaptoethanol) as described previously
(Bauerle et al., 1993). Protein concentrations were determined prior to
the addition of -mercaptoethanol by the BCA assay, and 40 µg of
protein was loaded per lane.
Proteins were separated on 12, 15, or 10 to 20% gradient gels and
electrotransferred to nitrocellulose membranes at a constant 12 V for
30 to 45 min at ambient temperature in a TransBlot semidry transfer
cell (Bio-Rad). Western immunoblot analysis was performed as described
before (Towbin et al., 1979). Blots were incubated with primary
antibodies for 2 to 4 h in TBS containing 0.1% Tween 20 plus 2%
nonfat dry milk at dilutions of 1:250, with constant rotation in a
hybridization incubator (LabLine, Melrose Park, IL) at 37°C. Alkaline
phosphatase-conjugated secondary antibodies were applied at a 1:5000
dilution.
Immunoblot data were quantified by scanning densitometry followed by
image analysis using a GS700 imaging densitometer and Molecular Analyst
2.1 software (Bio-Rad). Standard curves for molecular mass estimations
were generated by regression analysis of prestained marker proteins
using Molecular Analyst 2.1 software.
Preparation of Tonoplast Membrane Vesicles
Vacuolar membranes from red beet storage root were prepared as
previously described (Poole et al., 1984), frozen in liquid N2, and stored at 
80°C until use. ATPase and
proton-pumping activity were assayed after thawing according to
published methods (Poole et al., 1984). Membrane preparations typically
displayed specific activities for ATP hydrolysis in the range of 20 µmol mg
1 h1.
Tonoplast membrane vesicles were collected by centrifugation at
100,000g, dissolved in sample buffer, and heated to 95°C
for 5 min prior to SDS-PAGE. Protein loads were 20 to 40 µg per lane.
Fractionation of Tonoplast Vesicles
Tonoplast membranes equivalent to 400 µg of protein were thawed,
diluted into 1 mL of transport buffer (250 mM sorbitol, 100 mM KCl, and 25 mM BTP-Mes, pH 7.0), and then
collected by centrifugation for 30 min at 100,000g in a
fixed-angle TLA 120.2 rotor (Beckman). Membrane pellets were suspended
to 1 mg/mL protein in transport buffer and then incubated for 30 min on
ice in the presence of 5 mM MgSO4, 0 to 200 mM KNO3, and ±5
mM Tris-ATP. An aliquot was removed for measuring
proton-pumping activity, and the remaining mixture was centrifuged for
30 min to separate membrane and supernatant fractions. The membrane
pellet was washed once in transport buffer, and then membranes were
collected by centrifugation and resuspended in transport buffer. Wash
volumes were pooled and membrane and supernatant protein was
precipitated by adding TCA to 10% (v/v) and incubating for 45 min on
ice. Protein was pelleted by centrifugation for 15 min at 20,000 rpm in
a refrigerated microcentrifuge (Eppendorf). Protein pellets were
dissolved in 50 µL of sample buffer and heated to 95°C for 5 min
prior to SDS-PAGE.
Partial Purification of Red Beet H+-ATPase
Tonoplast membranes equivalent to 200 µg were thawed, diluted in
1 mL of transport buffer, and then collected by centrifugation as
described above. Tonoplast vesicles were solubilized with Triton X-100
as previously described (Parry et al., 1989). Briefly, vesicles were
resuspended in 0.70 mL of resuspension buffer (1.1 M
glycerol, 5 mM Tris-Mes, pH 8.0, 1 mM EDTA, 0.5 mM BHT, and 5 mM DTT), then slowly diluted by
dropwise addition of 0.75 mL of solubilization buffer (containing 8%
Triton X-100 and 4 mM MgSO4), and
stirred on ice. The resulting mixture was stirred gently for 20 min.
The detergent-solubilized mixture was partially purified by gel
filtration on Sephacryl S-400 as previously described (Parry et al.,
1989). A 60- × 0.75-cm-diameter column packed with Sephacryl S-400 was
preequilibrated with running buffer containing 10% glycerol, 0.3%
Triton X-100, 0.05 mg/mL phospholipid (Type IV-S, Sigma), 5 mM DTT, 1 mM Tris-EDTA, 4 mM
MgCl2, and 5 mM Tris-Mes, pH 8.0. The
entire sample volume was loaded and the column was run at a rate of 40 mL/h at 4°C. The detergent mixture was not centrifuged to remove
nonsolubilized membrane particles prior to loading. Typically, 40 1-mL
fractions were collected and 50-µL aliquots were assayed for ATPase
activity and protein concentration.
Protein Determination
The protein concentration of yeast extract was determined by BCA
assay according to the supplier's instructions (Pierce). The protein
concentration of tonoplast vesicle preparations and gel-filtration
fractions was determined by the method of Bradford (1987).
Determination of ATPase and Proton-Pumping Activity
ATPase activity was determined by measuring the amount of Pi
liberated from ATP at 37°C in a 20-min reaction using the Ames method
as previously described (Parry et al., 1989). Gel-filtration column
fractions were supplemented with sonicated 1.33-mg/mL type IV-S
phospholipid to preserve V-ATPase activity.
Proton-pumping activity in tonoplast vesicles was measured by the
method of Giannini et al. (1995). Typically, vesicles equivalent to 10 to 20 µg of protein were suspended in transport buffer containing 250 mM sorbitol, 50 mM KCl, 5 mM
MgSO4, and 5 µM acridine orange. The reaction was initiated by the addition of Tris-ATP to 5 mM final concentration, and proton pumping was monitored by
observing the decrease in acridine orange
A490 with a UV/visible light
spectrophotometer (model DU 640, Beckman) in "kinetics/time mode."
Rates were calculated from data collected at 10-s intervals during a
3-min reaction period.
Sequence Analysis
VMA6 homologous protein sequences were aligned using
Align Plus 2.0 from Scientific and Educational Software (State Line, PA). Alignment parameters were determined according to the work of
Myers and Miller (1988). Arabidopsis sequence fragments homologous to
yeast VMA6 were identified by a BLAST search of GenBank
(Gish and States, 1993). The following Arabidopsis fragments were used to generate a derived partial amino acid sequence: T13399, Z26026, Z24482, Z30468, T20646, H36140, H36163, H37538, T13974, R30209, T44170,
N97286, AA042689. The derived partial sequence reported in Figure 2 was
confirmed by identifying at least two overlapping fragments along the
entire length of the sequence.
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RESULTS |
A 44-kD Polypeptide from Red Beet Exhibits Immuno-Cross-Reactivity
with Yeast Vma6p and Cofractionates with Tonoplast V-ATPase
Immunoblot analysis with antiserum generated against the yeast
36-kD V0 subunit (
-Vma6p) consistently
revealed a single band in lanes containing red beet tonoplast protein
(Fig. 3, top). Immunoblot analysis using
the corresponding preimmune serum did not recognize either the yeast
36-kD subunit or the cross-reactive protein in tonoplast vesicle lanes
(Fig. 3, bottom). Neither -Vma6p nor preimmune sera recognized a
cross-reactive band in protein extracts from yeast vma6
mutant cells lacking the 36-kD subunit. Thus, the reactivity observed
in both yeast and red beet samples appears to be specifically due to
-Vma6p polyclonal antibodies present in the immune serum. The
molecular mass of the cross-reacting beet protein was estimated to be
44 kD by comparison with prestained molecular mass
protein standards in an adjacent lane.
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| Figure 3.
Immuno-cross-reactivity between yeast Vma6p and
beet 44-kD polypeptide. Proteins were separated on a 12% gel. Top,
Immunoblot with polyclonal antiserum prepared against yeast Vma6p
(Vma6p). Lane 1, Forty micrograms of whole cell protein from
wild-type yeast VMA6; lane 2, 40 µg of protein from
yeast vma6 mutant; and lane 3, 20 µg of red beet
tonoplast protein. Relative positions of molecular mass standards are
indicated. Bottom, Corresponding immunoblot with related preimmune
serum.
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To address whether the cross-reactive protein is specifically
associated with V-ATPase in tonoplast vesicles, we partially purified
the enzyme and monitored cofractionation of the cross-reactive protein
with peak V-ATPase fractions. Tonoplast vesicles were detergent
solubilized and proteins were separated by gel-filtration chromatography. The mixture was not centrifuged prior to loading on the
column, a step normally taken to remove any residual unsolubilized membranes. This allowed us to observe the distribution of the 44-kD
polypeptide between fully and partially solubilized fractions. Under
these conditions, solubilized V-ATPase eluted as a single broad peak
separated from bulk tonoplast protein, as evidenced by protein and
ATPase activity profiles (Fig. 4). ATPase
specific activity (corresponding to fractions 19-21 in Fig. 4) was
typically enriched greater than 10-fold in peak column fractions
compared with tonoplast vesicles (Table
II). A sharp early peak (fractions 13-16
in Fig. 4) with relatively lower ATPase specific activity corresponded to incompletely solubilized tonoplast vesicles eluting with the void volume.
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| Figure 4.
Gel-filtration purification of V-ATPase from
detergent-solubilized red beet tonoplasts. Tonoplast membranes were
detergent solubilized and proteins were separated by gel filtration as
described in ``Materials and Methods''. Equivalent aliquots of
even-numbered fractions were assayed for ATPase activity (micromoles
per hour of PO43 liberated) and total protein
concentration (milligrams) as described in ``Materials and Methods''.
 , Relative protein concentration;  , relative ATPase activity. Peak ATPase specific activity (fraction no. 20) was approximately 140 µmol mg1 h1, representing a 7-fold
enrichment.
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Table II.
Partial purification of V-ATPase from red beet
tonoplast membranes
V-ATPase was partially purified from detergent-solubilized tonoplast
vesicles by gel-filtration chromatography as described in ``Materials and Methods''. Equivalent aliquots of column fractions were assayed
for total protein and ATPase activity, and the fraction containing the
highest ATPase specific activity was identified. The results below
represent the average of three separate purifications.
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Column fractions were probed with polyclonal antiserum directed against
the 67-kD subunit of the red beet V-ATPase (-67 kD) to determine the
distribution of this peripheral V1 subunit.
Immunoblot results confirmed that the distribution of the 67-kD
V1 subunit closely correlated with V-ATPase
activity (Fig. 5, top). The 67-kD subunit
distributed between both the solubilized and unsolubilized V-ATPase
peaks. Very little of this V1 subunit was
observed in fractions containing the bulk of soluble tonoplast protein
released by detergent treatment (fraction no. 24), indicating that this subunit remained primarily associated with the enzyme complex during
column purification.
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| Figure 5.
Distribution of 67-kD subunit and 44-kD
polypeptide in gel-filtration fractions of detergent-solubilized beet
tonoplasts. Column fractions from the experiment described in Figure 4
were analyzed. Fraction numbers are indicated at the top. Arrows mark peak ATPase and protein fractions. The asterisk indicates the fraction
containing the peak ATPase specific activity. Top, Equivalent volumes
of each sample were separated on a 10 to 20% gradient gel and then
immunoblotted with anti-67-kD antiserum. A doublet band pattern was
typically observed with 67-kD antiserum when samples were separated
on gradient gels. Bottom, Same samples probed with Vma6p
antiserum.
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Distribution of the 44-kD cross-reactive protein was similar to that of
the 67-kD V1 peripheral subunit and closely
correlated with the observed ATPase activity peaks (Fig. 5, bottom). We
were unable to detect any of the 44-kD protein in peak fractions of soluble tonoplast protein. The 44-kD protein pattern correlated closely
with both the V-ATPase activity profile and the 67-kD peripheral
V1 subunit pattern. This suggests that the 44-kD
cross-reactive protein was tightly associated with the partially
purified V-ATPase fraction from detergent-solubilized tonoplast
membranes.
By comparing the RF against a standard curve
generated by regression analysis of prestained marker proteins, we
calculated the apparent molecular mass of the cross-reacting protein to
be 44.9 kD. This is within close range of 44- and 42-kD accessory subunits of beet V-ATPase previously described (Parry et al., 1989). To
determine whether the cross-reactive protein comigrated with one of
these previously reported accessory subunits, -Vma6p immunoblots
were compared with the protein band pattern of V-ATPase. Partially
purified V-ATPase protein fractions were separated by electrophoresis
and visualized directly by Coomassie staining or were transferred to
nitrocellulose and then stained with amido black to reveal the subunit
band pattern. Identical samples were transferred to nitrocellulose and
then immunoblotted with -Vma6p antibodies. The cross-reactive band
migrated closely with the prominent 44-kD subunit observed in partially
purified V-ATPase fractions (Fig. 6).
Thus, the previously described 44-kD accessory subunit of beet
V-ATPase appeared to be selectively immuno-cross-reactive with yeast
Vma6p antibodies.
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| Figure 6.
Comigration of cross-reactive band with 44-kD
accessory subunit of V-ATPase. Partially purified V-ATPase proteins
were separated on a 15% polyacrylamide gel, and then duplicate lanes
either were visualized by Coomassie staining (A) or were transferred to
nitrocellulose and immunoblotted with -Vma6p antiserum (B).
Coomassie-stained and immunoblotted band patterns were compared by
aligning band patterns of prestained marker proteins loaded in
adjacent lanes. Stained gel and developed immunoblots were digitally
imaged and analyzed using a scanning imaging densitometer.
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The 44-kD Polypeptide Is Dissociated from Tonoplast Membranes by
Urea Treatment
Treating yeast vacuolar membranes with urea quantitatively strips
Vma6p from the membrane, unlike other V0 subunits
that are integrally associated with the membrane (Bauerle et al.,
1993). To address whether the cross-reactive 44-kD polypeptide behaved in a similar fashion, we incubated isolated beet tonoplast vesicles in
transport buffer ± 8 M urea and examined the
distribution of the 44-kD polypeptide in supernatant and membrane
pellet fractions (Fig. 7). Similar to
yeast Vma6p, the 44-kD polypeptide was quantitatively removed from
membranes treated with 8 M urea. Peripheral
V1 57- and 67-kD subunits were also completely
dissociated from the membrane under these conditions (C. Magembe,
unpublished observations). Thus, the 44-kD polypeptide behaved like a
peripherally attached subunit in membrane fractionation experiments
with a strong chaotrope.
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| Figure 7.
Dissociation of 44-kD polypeptide from tonoplast
membranes by urea treatment. Tonoplast membranes equivalent to 50 µg
of protein were incubated in buffer alone (top) or buffer containing 8 M urea (bottom) for 30 min on ice. Membranes were collected
by centrifugation and protein samples from both membrane pellet and
supernatant fractions were prepared as described in ``Materials and Methods''. Proteins were separated on 12% gels and immunoblotted with
-Vma6p antiserum. Developed immunoblots were digitally imaged and
analyzed using a scanning imaging densitometer. S, Supernatant; P,
membrane pellet.
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KNO3 Treatment Inactivates V-ATPase and Causes
Partial Dissociation of 44- and 67-kD Polypeptides
Mild chaotropic agents such as KNO3 have
been shown to disrupt V-ATPase activity by causing specific
dissociation of V1 peripheral subunits from the
V0 sector (Ward et al., 1992). It is interesting that the dissociation effect is largely dependent on the presence of
MgATP and thus appears to be specifically correlated with the ATP-dependent active state of the holoenzyme. The use of such chaotropes can provide useful information about the relationship between V-ATPase activity and subunit assembly.
Figure 8 illustrates the effect of
KNO3 on proton pumping in tonoplast vesicles. The
ATP-dependent proton gradient was rapidly dissipated by addition of
gramicidin, confirming that tonoplast vesicles were tightly sealed
under incubation conditions. Proton-pumping activity was vanadate
insensitive (less than 15% inhibition in the presence of 200 µM vanadate), indicating that tonoplast vesicles were
relatively free of contaminating plasma membrane ATPase activity (not
shown). Proton-pumping activity was strongly inhibited by the presence
of 200 mM KNO3 in the transport
assay. Proton-pumping activity was similarly prevented by preincubating
tonoplast vesicles with 200 mM KNO3
plus MgATP prior to performing the assay. Following dilution of
preincubated vesicles into the assay medium, the resulting KNO3 concentration during the assay was
approximately 4 mM, well below the observed
Ki of 8 mM (refer to Fig. 10,
top). Thus, inhibition in this case was due to inactivation of the
V-ATPase during preincubation rather than inhibition during the assay.
The inhibitory effect of KNO3 preincubation was
much less pronounced in the absence of MgATP; preincubation with 200 mM KNO3 in the presence of either Mg2+ or Tris-ATP alone inhibited proton-pumping
activity by less than 50% (data not shown).
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| Figure 8.
Effect of KNO3 preincubation on proton
transport in beet tonoplast vesicles. Proton transport activity was
monitored as a percent decrease in acridine orange fluorescence
measured at 490 nm (% F). Tonoplast vesicles equivalent to 10 µg of
protein were diluted in 1 mL of transport buffer containing acridine
orange and equilibrated for 3 min. The reaction was started by adding 5 mM Tris-ATP, and absorbance measurements were collected
every 10 s for 3 min. At the end of the reaction Gramicidin D
(Gram) was added to a final concentration of 3 µM. a,
Nonpreincubated vesicles assayed in the absence of KNO3 for
3 min and then for 3 min after Gramicidin D addition; b,
nonpreincubated vesicles assayed in the presence of 200 mM
KNO3; c, vesicles preincubated with 5 mM MgATP;
and d, vesicles preincubated with 5 mM MgATP plus 200 mM KNO3.
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| Figure 10.
V-ATPase disruption as a function of
KNO3 concentration. Tonoplast vesicles equivalent to 50 µg of protein were preincubated with 5 mM MgATP and 0 to 200 mM KNO3 for 30 min on ice. Aliquots equivalent to 10 µg of protein were removed for proton transport assays in the presence of 5 mM MgATP. The remaining sample
was separated into membrane pellet and supernatant fractions and
processed as described for Figure 8. Bands corresponding to 67- and
44-kD polypeptides were quantified by imaging densitometry as described in ``Materials and Methods''. Top, Proton transport activity
following preincubation; middle, distribution of 67-kD polypeptide
between membrane pellet and supernatant fractions following
preincubation; bottom, distribution of 44-kD polypeptide following
preincubation.
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Dissociation of the 44-kD Polypeptide Is Dependent on ATP and
KNO3 Concentration
A series of membrane fractionation experiments were then conducted
using KNO3 to further examine the association of
the 44-kD polypeptide with tonoplast vesicles. Specifically, we sought
to correlate association of the 44-kD polypeptide with proton-pumping activity. Tonoplast vesicles were preincubated at 0°C with
KNO3 in the presence of MgATP, and then aliquots
equivalent to 20% of the total sample were assayed for proton
transport activity. The remaining membranes were pelleted, and both
membrane and supernatant fractions were examined for the 67-kD V-ATPase
subunit and the 44-kD polypeptide by immunoblot analysis. When compared
with the control, preincubation of tonoplast vesicles with 200 mM KNO3 plus MgATP resulted in
substantial dissociation of the 67-kD V1 subunit
(Fig. 9, top). The 44-kD polypeptide was
also partially removed from the membrane by preincubation with nitrate,
although to a lesser extent than the 67-kD peripheral subunit (Fig. 9, bottom).
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| Figure 9.
Dissociation of 67-kD subunit and 44-kD
polypeptide by MgATP and KNO3. Tonoplast vesicles
equivalent to 50 µg of protein were preincubated with 5 mM MgATP ± 200 mM KNO3 for 30 min on ice. Membranes were collected by centrifugation and protein
samples from both membrane pellet and supernatant fractions were
prepared as described in ``Materials and Methods''. Proteins were
separated on 15% gels and then immunoblotted. P, Membrane pellet; S,
supernatant. Top, Membrane pellet and supernatant fractions probed with
67-kD serum. Bottom, Same fractions probed with -Vma6p serum.
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Dissociation of both the 67-kD V1 subunit and the
44-kD polypeptide appeared to be a function of preincubation nitrate
concentration in the presence of MgATP (Fig.
10, middle and bottom). At the highest preincubation concentration of KNO3 tested (200 mM), 60% of the 67-kD subunit and 50% of the 44-kD
polypeptide were dissociated from the membrane. It is interesting that
complete inhibition of proton-pumping activity was achieved at much
lower concentrations, with an observed preincubation
Ki for nitrate inhibition of approximately 8 mM. Thus, although we consistently observed
concentration-dependent dissociation of V-ATPase subunits, it appeared
to be only generally correlated with loss of enzyme activity. In the
absence of ATP during preincubation, the observed
Ki for nitrate inhibition was approximately
260 mM. Some corresponding dissociation of V-ATPase subunits was observed; maximally, 25% of the 67-kD polypeptide and
10% of the 44-kD polypeptide were released during preincubation in the
absence of MgATP (not shown). However, the amount of subunit dissociation observed in the absence of MgATP was not dependent on
KNO3 concentration and therefore was not clearly
correlated with KNO3 effects on the active
V-ATPase holoenzyme.
| |
DISCUSSION |
In this report we present evidence supporting the identification
of a red beet protein homologous to Vma6p. The putative 44-kD homolog
was specifically cross-reactive with -Vma6p polyclonal antibodies,
indicating substantial sequence similarity with the yeast
V0 subunit. The 44-kD polypeptide cofractionated
with peak V-ATPase activity as well as the 67-kD subunit of the
V1 sector, indicating that it was tightly
associated with V-ATPase isolated from red beet. A 44-kD polypeptide
was previously reported to be associated with highly purified V-ATPase
preparations from red beet tonoplast (Parry et al., 1989). This protein
was released, along with other V1 subunits, by
cold inactivation conditions, leading the authors to conclude that it
is a peripheral V-ATPase subunit. Our results provide additional
support for the identification of this subunit and suggest further that
it shares significant similarity with a homologous subunit in yeast.
The existence of Vma6p homologs among animal, plant, and fungal species
suggests that this conserved subunit is important in the assembly and
function of V-ATPase holoenzyme.
The yeast 36-kD subunit is a tightly associated peripheral component of
the V0 sector of V-ATPase. Our results indicate
that the 44-kD unit is also peripherally attached and thus susceptible to removal with chaotropic agents such as KNO3.
KNO3 has been shown to inhibit V-ATPase activity
by dissociation of V1 subunits from the membrane
(Rea et al., 1987; Tu et al., 1987). At KNO3 concentrations sufficient to completely inhibit V-ATPase activity in
the presence of MgATP, neither the 67- nor the 44-kD subunit was
quantitatively released from the membrane. Thus, the release of
peripheral subunits may be a secondary consequence of a nitrate-induced conformational shift that leads to enzyme inactivation. Alternatively, some peripheral subunits may remain nonspecifically associated with the
tonoplast membrane. For instance, if tonoplast membrane preparations
contain a fraction of inside-out vesicles, then a portion of V-ATPase
would likely be protected from dissociation by nitrate (Rea et al.,
1987). Further biochemical studies are under way to determine the
membrane association of this putative Vma6p homolog.
Given the ubiquitous involvement of V-ATPase in acidifying internal,
and in some cases, external compartments, there has been much interest
in elucidating the mechanism(s) by which proton-pumping activity is
regulated. V-ATPase activity appears to be regulated in part by
posttranslational modifications of V1 subunits
(for reviews, see Forgac, 1996; Merzendorfer et al., 1997a, 1997b). In
addition, several reports have provided evidence supporting the
hypothesis that V-ATPase cellular activity may also be modulated by
regulated assembly-disassembly of V1 and
V0 sectors. For example, selective release of
peripheral V-ATPase subunits has been correlated with in vivo enzyme
inactivation in response to chilling in mung bean seedlings
(Matsuura-Endo et al., 1992). In Manduca sexta, regulation
of V-ATPase activity during larval development appears to be correlated
with a loss of V1 subunits (Sumner et al., 1995). Recently, Kane (1995) described in vivo assembly-disassembly of peripheral V-ATPase subunits in yeast cells in response to Glc deprivation. In yeast, active V-ATPase assembles by attachment of
preexisting V1 particles from a cytoplasmic pool
onto the V0 membrane sector. Hence, V-ATPase
components may be directly triggered to mediate assembly or disassembly
in response to intracellular signals. The positioning of Vma6p as a
peripherally associated V0 subunit required for
V1 attachment suggests a role in such a
regulatory mechanism.
We conclude that the 44-kD protein in red beet is a subunit of the
tonoplast V-ATPase holoenzyme and is homologous to a tightly associated
peripheral V0 subunit previously described in
yeast. Future studies will focus on a more detailed biochemical
characterization of this novel V0 subunit to
understand its role in V-ATPase assembly.
| |
FOOTNOTES |
1
This work was supported in part by funds from
the Hanna grant program (to C.B.) and a Lund Fund scholarship (to
C.M.).
2
Present address: The College of St. Catherine,
601 25th Avenue S., Minneapolis, MN 55454.
*
Corresponding author; e-mail cbauerle{at}piper.hamline.edu; fax
1-612-523-2620.
Received December 11, 1997;
accepted March 25, 1998.
| |
ABBREVIATIONS |
Abbreviations:
BCA, bicinchoninic acid.
BHT, butylated
hydroxytoluene.
V-ATPase, V-type proton-translocating ATPase.
| |
ACKNOWLEDGMENTS |
Portions of this project were completed during a sabbatical
leave by C.B in the laboratory of D.P.B. The authors thank Dr. Ron
Poole (McGill University, Montreal, Quebec, Canada) for providing antibodies against beet 57- and 67-kD subunits, Dr. Ben Lockhardt (University of Minnesota, Minneapolis) for use of his preparative ultracentrifuge, Lori Jahnke (Hamline University, St. Paul, MN) for
photographic assistance, and Dr. Sylvia Kerr for critically reading the
manuscript. The authors acknowledge helpful conversations with Dr.
Lynne Gildensoph (The College of St. Catherine, St. Paul, MN).
| |
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Abstract
Introduction
